The molecular pathogenesis of fully virulent, wild-type Y. pestis in relevant animal models has been relatively neglected because of the scarcity of secure BSL-3 facilities and trained personnel certified to work with this Class A select agent. The threat of bioterrorism and the emergence of multiply-antibiotic resistant strains of Y. pestis increases the urgency for a more detailed understanding of the host-pathogen relationship at the molecular level that may lead to the design of improved medical countermeasures and diagnostics. We have established mouse and rat models of bubonic plague that incorporate flea-to-rodent transmission to investigate the role of specific Y. pestis virulence factors and to characterize the host response to naturally acquired infection. We have characterized the kinetics, microbiology, and histopathology of bubonic plague in rats following intradermal injection of Y. pestis, and used this model to characterize the gene expression profile of Yersinia pestis in the infected lymph node during bubonic plague using whole-genome microarray technology. Our previous work has shown that three important Y. pestis virulence factors, Ail (a Y. pestis outer surface protein), the Type III secretion system (T3SS) encoded on the Yersinia virulence plasmid, and the plasminogen activator (Pla) encoded on the 9.5-kb Y. pestis-specific plasmid all act to limit the polymorphonuclear leukocyte response to bubonic plague infection in vivo (polymorphonuclear leukocytes, also referred to as PMNs or neutrophils, are phagocytic cells that are an important innate defense against infection). Thus, we now have several lines of evidence that the PMN response correlates with successful outcome to infection, and this aspect of host-pathogen interaction has become a focus of our lab. We discovered that the Y. pestis T3SS effector protein YopJ strongly inhibits the secretion of the IL-8 by human neutrophils. In addition, we reported that a small but significant percentage of Y. pestis, even T3SS-negative attenuated strains, survive and eventually replicate within phagosomes after being ingested by neutrophils. Infected neutrophils were also demonstrated to be taken up by macrophages in vitro, where Y. pestis could continue to replicate. The results indicate that neutrophil phagocytosis is not invariably fatal to Y. pestis, and that virulence factors other than the T3SS that counteract the strongly bactericidal environment of the neutrophil phagosome are important to plague pathogenesis. During the past year, we continued using intravital microscopy to image skin and skin-draining lymph nodes (dLN) early after infection with Y. pestis. Previous studies focused on the interactions between Y. pestis and host phagocytes (i.e. neutrophils, macrophages and dendritic cells) in nave mice. We have now extended these studies to include both actively and passively immunized mice and found dramatic increases in Y. pestis-neutrophil interactions in the skin and dLN in the presence of opsonizing antibody (Ab). We have also observed a large effect of opsonizing Ab on neutrophil extravasation into dLN tissue. We continue to work to identify mechanisms responsible for these phenomena to improve our understanding of Ab-mediated immunity to Y. pestis. We, and others, have reported that while the majority of Y. pestis taken up by neutrophils are quickly killed, approximately 10-15% of the bacteria survive and eventually replicate intracellularly. We continued investigating the mechanisms employed by Y. pestis to survive the highly microbicidal environment within a neutrophil. We used immunofluorescence microscopy techniques to differentiate between live and dead Y. pestis within human neutrophils. We found that phagosomes containing live bacteria remain partially immature and show reduced association with the primary neutrophil granule component neutrophil elastase and the active NADPH oxidase subunit p47phox. To further characterize the response of human neutrophils to Y. pestis in vitro, we measured surface expression of neutrophil activation and degranulation markers by flow cytometry, comparing Y. pestis to a variety of known neutrophil stimuli such as lipopolysaccharide, formylated peptides, PMA, and the Gram-positive pathogen S. aureus. Overall, the data showed that Y. pestis is less stimulatory towards neutrophils than S. aureus, inducing less surface expression of the primary and secondary granule markers CD63 and CD66b, respectively. These results suggest that Y. pestis possesses uncharacterized mechanisms to avoid or suppress neutrophil activation and phagosome maturation. To facilitate our studies involving in vitro assays of Y. pestis-neutrophil interactions we have established a system in the lab for the generation of immortalized murine neutrophil progenitor cells based on retroviral transduction of a Hoxb8-estrogen receptor construct in to bone marrow cells described by Wang et al. (ref: Nat Methods. 2006 Apr;3(4):287-93). We have successfully used this method to generate large numbers of murine neutrophils suitable for a variety of in vitro assays. We are currently working to characterize the response of murine neutrophils to Y. pestis and compare the results to those obtained in experiments using human neutrophils. We are also using murine neutrophils and macrophages to determine how the presence of opsonizing Ab affects the host cell response to Y. pestis in vitro. We continue to collaborate with Jason Cysters group at UCSF to determine the role of dLN subcapsular sinus macrophages (SCS Macs) in the pathogenesis of bubonic plague. Dr. Cyster provided CD169-DTR transgenic mice that allow for depletion of SCS Macs using diphtheria toxin treatment. Preliminary results showed a significant decrease in the Y. pestis CFU in the dLN of SCS Mac-depleted mice compared to controls. Work in the coming year will focus on confirming these preliminary results, gaining a better understanding of the fate of Y. pestis immediately after dissemination to the dLN, and determining whether or not SC Macs provide a protective niche for Y. pestis in this tissue. During FY2017 we also developed approved, arthropod containment level 3 (ACL-3) protocols to infect fleas with wild-type Y. pestis. We used an in vivo imaging system (IVIS) in conjunction with bioluminescent Y. pestis strains to monitor the incidence and dissemination patterns of infection in mice challenged by flea bite. Fleas were used within the first week after infection to evaluate what is known as early-phase transmission, and again 1-2 weeks after infection to evaluate a second, distinct transmission mechanism that is dependent on Y. pestis biofilm formation in the flea foregut. Results showed that flea-borne bubonic plague can follow an acute course, with dissemination from the flea bite site to the draining lymph node and systemic spread within a few days; or a prolonged course in which the bacteria multiply extensively in the intradermal flea bite site over several days, before either resolving or finally disseminating to cause systemic disease. This clinical picture differs from that seen following intradermal inoculation by needle, which invariably leads to acute disease. The chronic skin infection seen following flea bite challenge provides a new model to study immune responses in the dermis.